1. Field of the Invention
The present invention relates generally to histology, and, more particularly, it relates to the composition and use of a labelling complex capable of localizing cellular material present in tissue sections at very low concentrations.
2. Description of the Prior Art
The high binding affinity of biotin for avidin is the basis for a number of bioassays currently in use. It is known, for example, to incubate a biotinylated reagent with a prepared tissue section so that the reagent binds specifically to particular markers present in the tissue sample. By then exposing the tissue section to a labelling compound conjugated to avidin, for example, avidin conjugated to an enzyme or fluorescein, the markers are labelled and can be visualized by conventional methods.
A more sensitive method for localizing (or staining) antigens and other markers is that described in a brochure entitled "Lectins and Biotin/Avidin System" distributed by Vector Laboratories, Inc., of Burlingame, Calif. In that method, a primary antibody is "raised against" the antigen or other marker of interest. The primary antibody (e.g., rabbit IgG against human tumor antigen) is exposed to the prepared tissue specimens and will bind to the tumor antigen wherever present. The tissue sample is next exposed to a biotinylated secondary antibody (e.g., goat IgG raised against rabbit IgG) which results in the binding of two secondary antibodies to each primary antibody. Before addition, each secondary antibody derivatized with a number of biotin molecules which are available for binding with avidin. Thus, using these intermediate antibodies and a labelled avidin molecule and by exposing these reagents in sequence to the tissue sample, a label may be attached to the desired antigen site on the cell. Moreover, this method achieves an amplification in that a number of labelling molecules may be attached to each receptor site on the tissue sample. In this way, low concentrations of antigen may be visually detected.
It is further disclosed in the Vector brochure that rather than using an avidin conjugated label, the biotinylated secondary antibody may be exposed to unconjugated avidin. By then exposing the sample sequentially to a biotinylated label, additional avidin, and more biotinylated label, it is disclosed that an even greater amplification can be achieved. In practice, however, the use of a labelled avidin and the serial addition of avidin and biotinylated label, prove approximately equally sensitive, that is, they can detect equal threshold concentrations of marker sites on the tissue sample.
An alternate immunological staining technique in wide use which does not employ avidin-biotin is the peroxidase-antiperoxidase (PAP) technique. The tissue sample to be examined is first incubated with a primary reagent specific to the markers of interest. Thereafter, the sample is incubated with a secondary antibody raised against the primary reagent. The secondary antibody is present in excess so that the two binding sites of the secondary antibody are not exhausted by attachment to the immobilized primary. Antibody raised against peroxidase is obtained in the conventional manner and is incubated with peroxidase to form a peroxidase-antiperoxidase (PAP) complex. The PAP complex is then incubated with the tissue sample where it binds to the remaining sites on the secondary antibody. The sample is then treated with a reagent to cause a staining reaction with the peroxidase compound.
While the above-described staining techniques have proven successful in many applications, their sensitivity remains somewhat limited. If the method employing biotinylated primary reagent is considered to have an amplification of 1, the PAP technique and the methods employing biotinylated secondary antibodies have been found to have amplifications of approximately 3 and 5-8, respectively. It is desirable to provide a staining technique capable of an even greater amplification and of detecting antigens or other markers in tissue samples at even lower concentrations.